Insight into the molecular basis of breast cancer is needed to fulfill the goal of developing new, rational, and specific antineoplastic therapies. The recently identified novel protooncogene cyclin D1, or PRAD1, is strongly incriminated in breast cancer, and a variety of other human tumors including non-Hodgkins lymphoma, esophageal cancer, head and neck cancer, and parathyroid adenoma. Numerous studies, generally employing in vitro systems, have led to the hypothesis that cyclin D1 protein functions by activating cyclin-dependent kinase 4 (cdk4). This action is competitively inhibited by the p16INK4a tumor suppressor, and the retinoblastoma (pRB) tumor suppressor protein may be the primary substrate of cyclin D1-activated cdk4. The cyclin D1/cdk4/p16/RB pathway has tremendous potential significance to cancer, and may be targeted by mutation or regulatory derangements in most or possibly even all cancers. It is likely, however, that current concepts of cyclin D1 function will prove to be overly simplistic or incomplete when subjected to testing in highly complex in vivo systems, which are certain to be more relevant to human disease. An excellent model system for breast cancer investigation, and one well suited for testing interrelationships in the cyclin D1/cdk4/p16/RB pathway, is a recently developed transgenic mouse in which cyclin D1 overexpression is targeted to mammary tissue; this mouse develops preneoplastic changes and then mammary adenocarcinoma. This model also opens the door to testing the significance of other molecules, like the p53 tumor suppressor, in a manner highly relevant to breast cancer. The proposed studies are designed to (a) study the potentially synergistic influence of cyclin D1 and the p53 tumor suppressor gene, also implicated in breast cancer, by developing and characterizing mammary-targeted cyclin D1 transgenic mice that have one or two inactive p53 genes; (b) establish the relationship between cyclin D1 and RB derangements as synergistic or redundant, by developing and characterizing mammary-targeted cyclin D1 transgenic mice that have an inactive RB gene; and (c) examine relevance of cdk4 inhibition by p16 in mammary by developing and characterizing a transgenic mouse with mammary-targeted expression of a p16-insensitive dominant mutant form of cdk4. Features of in vivo oncogenesis to be characterized in these experiments include kinetics of tumor (and pre-neoplastic lesion) formation, histopathology, invasiveness/metastasis, p53 or RB gene loss of heterozygosity, genomic instability, subchromosomal localization of other cooperating genes, tumor cell growth rate, apoptotic index, and pRB phosphorylation. Overall, these results will carry important implications to the molecular pathogenesis of breast cancer, and ultimately contribute to novel treatment strategies.